In situ soil environment-based evaluation on degradation of biodegradable plastics

doi:10.21203/rs.3.rs-4818316/v1

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In situ soil environment-based evaluation on degradation of biodegradable plastics

https://doi.org/10.21203/rs.3.rs-4818316/v1

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The biodegradability of plastic is a critical factor in environmental sustainability. However, plastic degradation has been focused on closed systems via physical changes and CO₂ generation. We innovated a methodology on open system degradation in soil environments to reveal the authentic process of plastic degradation in nature. Polybutylene succinate (PBS), polybutylene adipate-co-terephthalate (PBAT), poly3-hydroxybutyrate-co-3-hydroxyvalerate (PHVB), and polylactic acid (PLA) were buried in a soil equipped with the lysimeter, the field applicable instrument that preserves and measures the in-situ soil conditions. Over two years, we tracked the soil electrical conductivity (EC), temperature, water content, and the plastic degradation products in the leachate−the monomers. The seasonal change in soil EC proved the plastic degradation, due to the decomposed plastic particles increasing the electrolyte concentration. The quantity of monomers increased over time, spiking during the summer months. A correlation was observed between the soil EC and monomer concentration. Despite the degradation-derived soil properties fluctuating with seasonal changes, the resilience of soils was maintained. Through long-term field experiments, we identified the seasonal degradation conditions of the actual soil environment and proposed a methodology of degradability that allows plastic targeting without disturbing the degradation media. These insights provide crucial knowledge for the biodegradable plastics market.

Earth and environmental sciences/Natural hazards

Earth and environmental sciences/Environmental sciences/Environmental chemistry/Geochemistry

Earth and environmental sciences/Environmental social sciences/Environmental impact

Earth and environmental sciences/Environmental social sciences/Sustainability

Earth and environmental sciences/Environmental sciences/Environmental chemistry/Environmental monitoring

Biodegradable plastics

Circular economy

UN SDGs

15. Life on land

Carbon cycle

Plastic waste

In the 2000s petro-based plastics production has continuously increased due to industrial growth, from 1.7 million tons in the 1950s to 300 million tons in 2015¹. Meanwhile, the global plastic circular economy policies encompass the solution for reducing the amount of plastic waste in the environment². The plastic waste caused severe environmental issues, with the cumulative amount in soils reaching 8 billion tons^3,4. Therefore, the United Nations Environment Programme (UNEP) has signed the 'Plastic Treaty' to curb plastic pollution. Thus, biodegradable plastics have gained attention as an alternative solution to these environmental issues⁵. Once the biodegradable plastics return to the soil at the end of the life cycle, they undergo a relatively rapid decomposition process. The observation of the degradation is possible within a few years⁶. Meanwhile, conventional petroleum-based plastics take hundreds of years to decompose^7-9. A long period of experiments is required to observe the behavior and changes during the degradation in the soil when the waste plastics are buried in¹⁰.

The degradability of biodegradable plastics has been proven, but limited to ex-situ methodologies in closed systems where the temperature, air flux, and moisture content are controlled^11-14. The standard methods use an ideal design by capturing carbon dioxide as the evidence of degradation from the biodegradable plastic incubation^15-17. The ISO standard specifies a methodology for testing compost, sediment, and soil biodegradability under aerobic and anaerobic conditions¹⁸, but only under closed and controlled conditions¹⁶. However, there is a limitation in capturing all the carbon dioxide generated during the test without loss, leading to potential errors. This presents a significant obstacle for application in in-situ environments. Furthermore, because all biodegradable plastics release CO2 regardless of type, it is impossible to track specific plastic types. The degradability test also relies on physical changes, such as morphological deterioration and weight loss of the biodegradable plastics¹⁹^,²⁰. However, these physical measurements inevitably disturb the degradation media, making long-term continuous tracking difficult.

To understand the decomposition rate and degree of decomposition of each plastic when various plastics are mixed in landfills or perennial farmland, it is necessary to go beyond comprehensive carbon dioxide emissions and trace the specific indicators. Therefore we estimated the degradability of biodegradable plastics with a real environment-applicable method that overcomes the previous limitation. We revealed the changes in soil properties derived from the degradation of biodegradable plastics which was a limitation in previous studies^21,22. To preserve the in-situ environmental conditions, the soil changes were observed by installing a lysimeter, which is a field-applicable instrument that can trace the substances in soil²³ (Fig.1). It has been used to monitor the physicochemical properties in soils impacted by crop, horticulture, landfills, and animal carcass disposal²⁴. Though the lysimeter has a long history in soil research, plastic, which is one of the significant environmental concerns of the current generation, has not been adopted yet. Utilizing a lysimeter to directly confirm the influence of numerous biotic and abiotic factors in the soil environment²⁵, we possibly detected the plastic degradation products flowing into soil water and the leachate.

This study suggests a novel approach to identifying the degree of biodegradation in natural soil media, ultimately taking a step closer to a sustainable environment. It presents the possibility of simulating the real environmental conditions in which biodegradable plastic-based wastes circulate as natural elements in the soil. Also, the pioneering method quantitatively estimates the degradation of biodegradable plastics and enables qualitative analysis of plastic-specific degradation even under mixed plastic conditions. It is possible to verify a clear degradation process and provide basic knowledge that guarantees the reliability of the rapidly growing biodegradable plastic market.

Natural soil degradation condition

We investigated not only the degradation of biodegradable plastics in a natural open system but also the condition of the degradation media (Fig. 2). The temperature and water content of soil media were observed for 30 months during the plastic degradation, which trends were statistically identical between inside (lysimeter) and outside (background) of the incubation barrier (Fig. S1).

The climate in South Korea has four seasons such as summer, fall, winter, and spring, with different temperatures and humidity. The annual temperature ranges from 36.1°C at the highest in summer to -27.3°C at the lowest in winter and the atmospheric humidity varies between 30~70%. Plastic degradation in natural environment conditions occurs within these ranges (Fig. S2). However, soils in the ecosystem have a buffer capacity to maintain the resilience of the physicochemical properties. The condition of soil media where the actual plastic degradation occurs is different from what the weather information indicates. The soil temperature ranged from 28.5 °C to 0.6 °C in different seasons, while the water content maintained a steady range of 0.2 to 0.4 m³ m^-3 (Fig. 2A).

We statistically calculated the degradation condition by the four seasons. The seasons were classified every three months starting from June (Table S1), which represents the summer (June to August), fall (September to November), winter (December to February), and spring (March to May). Fig. 2B encapsulates the dynamics of soil temperature and moisture content across different seasons. Soil temperature provided a significant impact on the biodegradation of plastics causing microbial activities to break down biodegradable plastics into monomers. During the summer period forming a microbial activity-friendly environment, the organic matter including biodegraded monomers was produced²⁶. However, as the soil temperature dropped during winter, the environmental condition for microbial activities became unfavorable resulting in the lowest values of soil EC and the least amount of monomer produced.

In summer, the soil temperature is relatively high and stable, with an average of around 25°C, conducive to robust microbial activity which is essential for the biodegradation process. The soil water content is consistently near 0.3 m³ m^-3, which suggests an optimal hydric state for microbial enzymatic activity and the sustenance of microbial communities responsible for plastic degradation. In the case of fall, there is a noticeable decline in soil temperature, potentially leading to a moderated biodegradation rate as microbial metabolism slows. Despite this, the soil moisture content remains largely unaffected, averting the compounding negative impact on biodegradation that would accompany drier conditions. The monitored soil condition can be implicated in the biodegradation of bioplastics in actual environments.

Conversely, the marked reduction in soil temperature recorded during winter, maintaining an average below 15 °C, is likely to substantially decrease microbial degradation activity. However, the persistence of soil moisture content at 0.3 m³ m^-3throughout the season implies that moisture-related limitations to microbial activity are minimized, which may partially offset the temperature-induced reduction in biodegradation rate. The progressive increase in soil temperature observed in spring augurs well for the reactivation of microbial degradation processes after the winter slump. The sustained optimal moisture content further supports this reinvigoration, ensuring that the biodegradation process does not face hydric constraints.

The soil temperature is the primary seasonal driver that could impact the biodegradation in soil, with variations between seasons. In contrast, soil moisture content remains relatively constant, indicating that water availability is unlikely to be a variable constraint throughout the year. The resilience of soil moisture content amidst seasonal temperature fluctuations provided a stable medium for biodegradable plastic degradation. We identified the seasonal patterns of soil temperature and moisture, which are critical for the strategic planning of bioplastic waste management and the development of biodegradation models in natural soil conditions.

The indicator of degradation in soil

During the degradation progresses, the polymers are broken down, and the monomers are produced and mixed with the soil water. The monomer analysis demonstrated a pattern similar to that of soil EC. The leachate from the soil without biodegradable plastic did not contain monomers.

Driven by the degradation of biodegradable plastic, the soil electrical conductivity (EC) fluctuated (Fig. 3). The EC of the soil in the lysimeter showed a significantly different trend from that of the background soil in specific seasons. The soil EC refers to the number of salts in the soil, and it varies with the acidity and the number of displaceable ions²⁷. For the whole experimental period, soil EC in the background maintained an average of 0.61044±0.06511 dS m^-1 without significant differences among the seasons. The soil was non-saline (less than 2 dS m^-1) and in the average range of Korean upland soil²⁸. However, the EC of the soil in the lysimeter drastically increased in summer and fall. While the considerable increase occurred from June to November annually, the winter and spring remained the parallel trend with the background soil.

The strikes of soil EC in the summer and fall seasons gradually reduced by the year. Significant difference lasted for the first two years, and it converged to the degree of background soil in 3^rd year (Fig. 3). The first summer recorded a maximum of 3.646 dS m^-1 which is 450% higher than that of the background control soil (lysimeter maximum: 3.646 dS m^-1; control soil maximum: 0.812 dS m^-1, difference of 2.834 dS m^-1). Subsequently, after the parallel maintenance of EC in winter and spring, the difference rose to approximately 320%, with a maximum of 2.196 dS m^-1 in the lysimeter soil.

The change in soil EC can occur due to the degradation of plastics since soil media provides a suitable condition for hydrolysis. Enough water can be preserved by the water-holding capacity of soils and keep reacting on the plastic particle surfaces. Hydrolysis-derived degradation of plastic generates the organic acid substances, and additional charges from the products contribute to the increase of soil EC. The position of the soil sensor in the reservoir (63.5 cm) of the lysimeter was at the mid-depth. Thus, the content of organic acids and EC can be higher when it is accumulated in deeper soil.

The biodegradable plastics mixed with the soil in the lysimeter were actively degraded under anaerobic conditions, and the most active degradation that alters soil EC occurred in the initial period. Considering that biodegradable plastic degradation occurs in categorical procedures, such as biodeterioration, bio-assimilation, and mineralization, hydrolysis acts as a trigger to facilitate the subsequent biological degradation. Once the early phase of degradation (hydrolysis) is accomplished, the reduction of soil EC indicates the succeeding bacteria-based degradation will enhance.

The soil leachate was expected to contain biodegradation products, and we analyzed the monomers in it qualitatively and quantitatively. Since the monomers determine the biodegradability of plastics, it is possible to estimate the biodegradation more accurately and plastic specifically. When multiple types of plastics are mixed like in a real landfill, it is necessary to track the degradation of specific monomers rather than count on the comprehensive carbon dioxide emissions. The target substances were succinic acid (SA) and 1, 4-butanediol (BD) for poly(butylene succinate) (PBS), adipic acid (AA) and BD for poly(butylene adipate-co-terephthalate) (PBAT), lactic acid (LA) for poly(lactic acid), and 3-hydroxybutyric acid (3-HBA) for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHVB).

The presence of monomers in leachate is the outcome of the inflow of decomposition products including organic acid substances in the soil into the soil water. As shown in Figure 4, the number of monomers detected was different for each kind of biodegradable plastic, but the elution of monomers showed similar patterns. From immediately after the incubation, the amount of monomer detected increased. However, from fall to winter and spring seasons, the number of monomers hardly changed. This was the result of the decrease in the activity of microorganisms involved in the degradation of biodegradable plastics during the winter period when relatively low temperatures were maintained. In the summer and fall of the second year when soil temperature rose again, the detected number of monomers increased back. However, compared to the first year, the number of monomers detected in the summer and fall of the second year decreased. The number of monomers demonstrated a pattern similar to that of soil EC. That is, the change in soil EC is expected to be the result of the degradation of biodegradable plastics, and this was verified through qualitative and quantitative analysis of monomers in leachate.

Among the target monomers, HBA was detected at the highest concentration, followed by SA, BD, and AA in that order. This result suggested that the degradation of PHA in soil occurred at a faster rate than in other biodegradable plastics. In the case of PLA, a degradation product, LA, was not detected in the first year but started to be detected during the last sampling period in November 2022. These trends can be unaligned with the previous plastic biodegradation test results in a closed system because industrial composting conditions are a lot different from natural ones which needs in-depth further research.

Validation of Plastic Degradation and Soil Safety

The degradation of biodegradable plastics in the soil was confirmed through total organic carbon (TOC) analysis of leachate. The biodegradation analysis method of aerobic plastics set by the Organization for Economic Cooperation and Development (OECD 301B)²⁹ or the International Organization for Standardization (ISO 22404)³⁰ measures the amount of carbon dioxide produced during decomposition. Since the standards are made in a closed system that controls the environmental conditions necessary for the biodegradation of plastics, it is assumed that the source of carbon dioxide only arises from the biodegradation of plastics. This study was carried out under the open system, but it was highly possible that the carbon dioxide generated because of the degradation of biodegradable plastic flowed into soil water and changed the amount of TOC. Moreover, ISO 22404 states that the amount of carbon dioxide can be indirectly measured through TOC analysis. As a result, it was confirmed that the TOC values of leachate had a similar tendency to the soil EC. (Fig. 4).

We also analyzed the leachate sample ("210706") with the highest values of EC, rate of monomer content increase, and the TOC using an inductively coupled plasma mass spectrometer (ICP-MS) to measure elements qualitatively and quantitatively (Fig. S5). There are two possible explanations for the elevated levels of Mn. First, the bare land used in this study was previously a vegetation cycling field, which could have resulted in an increase in magnesium and manganese due to nutrient uptake and photosynthesis³¹. Second, Mn-oxides present in the soil may catalyze the breakdown of organic matter into smaller compounds through oxidation^32,33, potentially producing the monomers from biodegradable plastics and contributing to an increase in TOC. However, more detailed and targeted analyses are needed to draw definitive observations.

Territorial toxicity was determined through the germination tests using two different soil leachate samples: "210706" and "220506" (least amount of monomers and EC values). The seed germination rate of "210706" was 0 % but recovered up to 92 % a year later with self-purification of soil through microbial activities. On the other hand, that of "220506" was not significantly different from the control sample. Moreover, to determine if a specific monomer could affect seed germination, individual monomers that were detected in the soil leachate were investigated as well. As a result, AA presented the lowest EC₅₀ and significant difference at the solution of 3mg 100mL^-1. On the other hand, SA and BD did not affect seed germination at the concentration detected in soil leachate. (Fig. 5 and Table S4, S5).

In addition, the TOC and seed germination in soil leachate presented significant differences from the control samples. The results of TOC indicated that the production of carbon sources was more fulfilled through plastic biodegradation, and the biodegradation directly affected the TOC values in leachate. Moreover, through the seed germination test, it was determined that specific monomers may cause adversity to plant growth in the short term, but the soil environment can still recover with it through microbial activities. The highly produced organic acids derived from the biodegradation of plastics, on the other hand, could promote soil aggregation and increase microbial activity, thereby improving soil quality. Therefore, a longer term of monitoring and analysis will be necessary to draw verified conclusions.

Prospective Applications

The ultimate purpose of biodegradable plastics is to simultaneously cultivate land and biodegrade plastics. Beyond simply understanding that biodegradable plastics degrade under natural conditions, continuous research is needed to investigate how the byproducts of plastic degradation affect plant growth. It is currently in the limelight and corresponds to the goals pursued by environmental, social, and governance (ESG) issues or sustainable development goals (SDGs).

Acknowledgment

This study was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2021R1A2C2011734) and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2021R1A6A1A10045235). This work was also supported by the Ministry of Science, ICT & Future Planning (NRF-2021M3H4A3A02102349).

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Soil (media) information

Bare land is in Korea University Deokso farm at Deokso-ri, Wabu-eup, Namyangju, Gyeonggi-do, Korea (37°34’56"N 127°14’04"E). Iron fences were installed on the side of the bare land to prevent the invasion of wild animals, and no other facilities were installed to avoid environmental disturbance. Weed control of the lysimeter site was periodically performed to maintain the bare topsoil to avoid hindrance of the vegetation. The bulk density was 1.26 g cm^-3in the range of general Korean soils. The soil pH is 6.8 and electrical conductivity (EC) was 0.019 dS m^-1, containing 9.6 mg kg^-1 of available phosphorus and 6.8cmol⁺ kg^-1 of exchangeable cations. The total nitrogen content of the soil was 0.056%, and it contained 1.9% of the soil organic carbon. The C/N of experimental soil in the natural environment was 33.75.

Biodegradable plastic preparation

PBS, PBAT, PHA, and PLA³⁴ were selected due to their generality in the biodegradable plastics market. The mechanical properties of these plastics are shown in Table S2.

For both qualitative and quantitative detection of biodegradable plastic monomers using the HPLC-MS system, methanol (MeOH), acetonitrile (ACN), and water, all purchased from Fisher Scientific and of optima grade, were used as the mobile phase solvents. Reference standards for LA (for PLA), SA (for PBS), 1,4-BD (for PBS and PBAT), and AA (for PBAT) were obtained from Sigma-Aldrich. PHBV (MW: approx. 10,000) was sourced from CJ (South Korea) to be depolymerized into 3-hydroxybutyric acid (3HBA). Monomer solutions (SA, BD, AA, and LA) were mixed and diluted for instrumental optimization and method validation.

For PHBV monomers, 3HBA was chosen as the main target due to its prominence among over 91 PHA constituents, including PHBV³⁵. Therefore, unlike SA, BD, AA, and LA, PHBV monomer standards had to be created in-house. Common depolymerization methods for HBA include methanolysis and hydrothermal methods, with methanolysis being selected for its higher monomer recovery rate and safety. We adopted the depolymerization method described by Parodi et al.³⁶ using PHBV, a PHA produced by various microorganisms³⁷. The PHBV polymer was synthesized by methanotrophic bacteria using omega-hydroxyalkanoate co-substrates³⁸. 500 mg of PHBV was immersed in the mixture solvent of 3.53 mL of MeOH and 1.55 μL of H₂SO₄. For the depolymerization, 500 mg of PHBV was immersed in a solvent mixture of 3.53 mL MeOH and 1.55 μL H₂SO₄, stirred, and heated for 7 hours at 140°C. After cooling to room temperature, 1.163 mg of NaOH, equivalent to the molar amount of H₂SO₄, was added and stirred for 15 minutes at room temperature. Methyl 3-hydroxybutyrate (MHB) was then recovered through reduced pressure distillation at 75-80 °C. With a yield of over 95%, the amount of MHB was considered equivalent to the amount of 3HBA due to depolymerization. NMR analysis confirmed the presence and structure of MHB (Fig. S4). The developed MHB solution was subsequently applied to the optimized method using the monomer mixture solutions.

Field incubation design

Korea experiences distinct seasonal temperature variations, with Namyangju, the location of the field study, averaging 24.0 °C in summer and -2.0 °C in winter (KMA Weather Data Service). Bare land soil exposed to these weather changes undergoes a seasonal freeze-thaw cycle of pore water, leading to physical, chemical, and biological transformations. To monitor meteorological changes, atmospheric temperature, humidity, and precipitation were recorded at 15-minute intervals using ATMOS 14 and ECRN-100 sensors throughout the experimental period.

The Drain Gauge G3 Lysimeter from Meter Environment (USA) was used in the study. This reservoir (d: 25.4cm, H: 63.5cm) holds approximately 32,000 cm³ of soil, with an open top to allow soil water to pass through to the underground reservoir. The reservoir contained soil mixed with biodegradable plastics, and gravity water leached through the pores, accumulating in the drainage at the bottom. When enough leachate was collected, it was pumped out using a peristaltic pump (METER Group ECRN-100). A sensor in the drainage monitored water level, temperature, and electrical conductivity (EC) (Fig. 2C). Additional sensors monitored soil conditions inside and outside the lysimeter.

During lysimeter installation, field soil was excavated to a depth of 2 meters, exposing the organic matter layer (O layer), A layer, and B layer. The lysimeter was buried at the depth of the A layer, and soil from this layer was used to fill it. The soil at the experimental site is sandy loam, composed of sand (64.6%), silt (29.7%), and clay (5.7%), with a density of 1.1 to 1.2 g m^-3, typical of basic farmland soil in Korea. Although this soil has been agricultural land for decades, it has lain fallow for the past five years.

The reservoir was filled with soil mixed with biodegradable plastic at a 12:1 ratio, following the biodegradable plastic degradation test method (ISO-14855). Approximately 32 kg of A-layer soil and 0.67 kg of each type of plastic (a total of 2.6 kg) were used. Pure polymer resin particles without additives were used for all plastics in the experiment, mixed in a 1:1:1:1 weight ratio. Soil and plastic particles were thoroughly mixed to promote decomposition. The reservoir was installed about 15 cm below the topsoil, and the topsoil was compacted to its original density.

Only the natural precipitation and atmospheric moisture contributed to the water source. Leachate collected seasonally from the reservoir was gathered at low temperatures without exposure to oxygen and sunlight. The volume of leachate was highest in the summer rainy season due to high precipitation, whereas no samples could be collected in winter (December to February) because of low precipitation and minimal soil gravity water movement due to low temperatures. Sampling was conducted in spring and fall when moderate precipitation allowed sufficient leachate collection.

Monomer quantification

Monomer identification and quantification for assessing the degradability of biodegradable plastics were conducted using an HPLC system (1260 Infinity, Agilent) combined with a single quadrupole mass spectrometer (6120, Agilent). The analysis was performed in both positive mode (for PBS, PBAT, and PHBV) and negative mode (for PLA)³⁴. The separation was carried out with a Luna Omega PS C18 100A column (150×4.6 mm×5µm), maintaining the column temperature at 25 °C and the flow rate at 0.1 mL min^-1. The injection volume was 10 μL, and the total run time was 40 minutes (37 minutes for the run and 3 minutes post-run). The mass spectrometer operated in electron impact mode at 70 eV, with a mass range of 30 to 1000 m/z. Additionally, solvent composition, spray voltage, nebulizing pressure, drying gas flow, and drying gas temperature were optimized using target monomers to establish optimal conditions for qualitative and quantitative detection of the analytes (SA, BD, AA, and LA), improving sensitivity for further applications. The optimal settings were determined to be ACN:water/5:5 of solvent composition, 1500 V of spray voltage, 50 psi of nebulizing pressure, 12 min L^-1 of drying gas flow, and 290 °C of drying gas temperature.

Calibration curves (linearity), the limit of detection (LOD), the limit of quantification (LOQ), accuracy, and precision were established for qualitative and quantitative analyses. Calibration curves were created by running different concentrations of standard solutions diluted from a 1000 ppm stock mixture of SA and BD (mimicking degradation from PBS), AA and BD (from PBAT), LA (from PLA), and 3-HBA (from PHA) in simulated soil water. The LOD and LOQ were determined based on these curves. Standard solutions were prepared at concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, and 0.4 μg in simulated soil water. Each calibration standard was analyzed in triplicate, and the mean values (area size) were plotted against the concentrations of the target analytes. Calibration curves for each analyte, along with their respective LOD and LOQ values, were shown in Fig. S3.

Precision and accuracy were evaluated by analyzing monomer standards at three quality control (QC) levels: low (0.05 μg), medium (0.2 μg), and high (0.4 μg), with each level tested in triplicate. Intra-day precision (repeatability) was assessed within a single day, while inter-day precision (reproducibility) was assessed over three different days. Precision results were expressed as the relative standard deviation (RSD), with values up to 20% deemed acceptable per Karnes and March et al.³⁹. Accuracy was considered acceptable if measurements fell between 80 and 120%³⁹. The RSD for triplicates at all three QC levels ranged from 1 to 17% for both intra-day and inter-day precision. Accuracy at the three QC levels ranged from 83 to 120% (Table S3). Thus, the RSDs for both intra-day and inter-day precision, along with accuracy values at all QC levels, were within the acceptable range.

Toxicity Test

A seed germination test was performed to evaluate the terrestrial toxicity of soil leachate, as leachate can impact plant growth. The procedure was based on a modified version of the OECD 208 standard, tailored for this study by omitting soil application⁴⁰. In this test, a petri dish was lined with filter paper and moistened with 5 mL of soil leachate and monomer mixtures dissolved in distilled water, which served as control groups. Thirty radish (Raphanus sativus) seeds were placed on the moistened filter paper, then the dish was covered with a lid and sealed with Parafilm to prevent evaporation. The plates were incubated for 5 days at 25 °C. After incubation, the germination rate (GR) and radicle length (RL) were measured. Germination was defined as both the plumule and radicle reaching lengths of over 2 cm.

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In situ soil environment-based evaluation on degradation of biodegradable plastics

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Natural soil degradation condition

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